APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes

The interaction of Plasmodium falciparum—infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or “knobs” on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer’s clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

The plasmids pCC1-KAHRP-FLAG-APEX2 and pDC2-U6-Cas9-gRNA kahrp were co-transfected into the P. falciparum 3D7 strain to generate a transgenic strain in which FLAG and APEX2 tags were inserted at the 3′ end of the endogenous kahrp gene, creating a chimeric KAHRP-FLAG-APEX2 gene (Fig. 1A).The first transgenic parasites were observed after 21 days under drug pressure.Diagnostic PCR confirmed integration of the cassette to the endogenous KAHRP locus-amplicons from the primers PS110/PS102 and PS103/PS114 of 1.1 kb and 1.6 kb, respectively, confirming correct integration (Fig. 1B).
To test the ability of the integrated APEX2 enzyme to catalyse biotinylation, enriched late-stage KAHRP-FLAG-APEX2 parasites were incubated for 30 min in the presence or absence of Biotin-Phenol (BP+ /−), followed by 1 min of oxidising in the presence or absence of H 2 O 2 .Samples were therefore created from four conditions: BP−/H 2 O 2 − (both without BP and H 2 O 2 ); BP−/H 2 O 2 + (without BP and in the presence of H 2 O 2 ); BP+/H 2 O 2 − (in the presence of BP and without H 2 O 2 ); and BP+/H 2 O 2 + (both in the presence of BP and H 2 O 2 )-biotinylation is only expected in the presence of both BP and H 2 O 2 .After quenching, iRBCs were solubilised using saponin treatment, which disrupts the RBC membrane but not the parasite membrane, allowing release of the RBC cytosol with its high levels of haemoglobin.The saponin-treated pellet, which contains both parasites and membrane fragments from the iRBC such as Maurer's Clefts, was then solubilised using RIPA buffer (RIPA-soluble proteins) and separated by NuPAGE.
Anti-FLAG tag antibodies confirmed the expression of the chimeric protein KAHRP-FLAG-APEX2 in all conditions (Fig. 2A), although the chimeric protein ran higher than predicted molecular mass of 100 kDa, most likely due to charged residues in the C-terminus of KAHRP, or the histidine-sequence repeats, as similar repeats are well-known to produce atypical migration of other proteins in P. falciparum 31 .Multiple bands of smaller size were also detected, suggesting partial proteolytic cleavage.It is not known whether this occurred within the parasite or post-lysis, although it should be noted that proteolytic processing is a common feature in P. falciparum proteins.Wildtype P. falciparum 3D7 strain parasites and a P. falciparum transfectant strain in which FLAG-APEX2 was expressed from an episome (Fig. S1A), unlinked to any endogenous protein, were used as controls and show clearly different sized products in Western blot (Fig. S1B), confirming the bands in Fig. 2A are specific to the presence of KAHRP-FLAG-APEX2.
To test the efficacy of the APEX2 biotinylation assay, RIPA-soluble proteins were probed with streptavidin-HRP to label biotinylated proteins.Only the condition in which both the BP substrate and the oxidizing agent H 2 O 2 were present revealed biotinylated proteins, with multiple bands over a large molecular weight range, suggesting biotinylation of multiple proteins (Fig. 2A).In the episome-based FLAG-APEX2 line, biotinylated proteins were also observed by streptavidin-HRP blot only in the presence of BP and H 2 O 2 (Fig. S1B).No signal was observed in the negative controls, in which either BP or H 2 O 2 were omitted in the KAHRP-FLAG-APEX2 line (Fig. 2A) or in wildtype 3D7 parasites (Fig. S1B).Biotinylated proteins were enriched using streptavidin-coated  www.nature.com/scientificreports/beads from all KAHRP-FLAG-APEX2 samples from all four conditions-again, protein material were observed only in the lane containing the sample treated with both BP and H 2 O 2 (Fig. 2B).This establishes that APEX2tagged KAHRP catalyses biotinylation of proteins within the parasite, and that this biotinylation is dependent on incubation with H 2 O 2 and the BP substrate.

Localisation of KAHRP-FLAG-APEX2 and biotinylated proteins by confocal immunofluorescence
Immunofluorescence assays (IFA) using anti-FLAG antibodies showed a punctate distribution of KAHRP-FLAG-APEX2 inside the iRBC cytoplasm (Fig. 3A, grey arrows), consistent with previous reports of KAHRP location 32 .Staining was also visible inside the parasite near the nucleus, potentially representing KAHRP that was being synthesised, however, similar staining was also present with anti-FLAG antibodies in wild-type parasites, suggesting the labelling is non-specific (Fig. 3A).By contrast the punctate distribution of anti-FLAG staining was absent from 3D7 parasites, suggesting that this localisation inside the iRBC cytoplasm is specific to the expression of KAHRP-FLAG-APEX2.
To establish the localisation of proteins biotinylated by KAHRP-FLAG-APEX2, we used IFA and fluorescentlytagged streptavidin (streptavidin AF488) to detect proteins that were labelled after incubation with H 2 O 2 and the BP substrate.Structures within the iRBC cytoplasm were present in schizonts treated with BP+/H 2 O 2 +, but not schizonts from which H 2 O 2 was omitted, confirming that the labelling was dependent on APEX2 activity (Fig. 3B).Co-localisation of streptavidin-AF488 with antibodies against membrane-associated histidine-rich protein (MAHRP), a specific marker of MC known to colocalize with KAHRP [33][34][35] , showed that proteins biotinylated by KAHRP-FLAG-APEX2 were partially co-localised to the MC (Fig. 3C, white arrows).Conversely, IFAs using streptavidin-AF488 with the parasite line expressing episome-based FLAG-APEX2 showed biotinylated proteins were primarily localised to the cytosol, and did not co-localise with anti-MAHRP (Fig. S2).In this parasite line, a partial co-localization between biotinylated proteins and the Golgi apparatus at the perinuclear regions of P. falciparum segmented schizonts was observed using an antibody raised against the ERD2 [36][37][38][39] (Fig. S2).

KAHRP-FLAG-APEX2 parasites produce iRBCs that display knobs and bind to human endothelial cells
To confirm that the addition of the FLAG-APEX2 tags did not affect KAHRP function, the presence of knobs in the trophozoite/schizont-iRBCs of the transfectant line was examined by scanning electron microscopy (SEM) compared to the 3D7 parental strain and uninfected RBCs.SEM showed a typical knobbed surface on both 3D7 and transfectant trophozoite/schizont-iRBCs (Fig. 4).This pattern was consistent with previous studies 15,18,31 , indicating that KAHRP-FLAG-APEX2 is correctly expressed and can support the production of knob structures in the iRBC membrane.To test for any impact on cytoadherence, we incubated human umbilical vein endothelial cells (hUVECs) with both 3D7 and transfectant trophozoite/schizont-iRBCs, and saw adherence of iRBCs from both strains, whereas no adherence was seen when hUVECs were exposed uninfected RBCs (Fig. S3A-C) 40 .There was no statistical difference in the binding frequency of iRBCs from 3D7 and KAHRP-FLAG-APEX2 transfectant lines to hUVECs (Fig. S3D).Together with the SEM data, this suggests addition of the FLAG-APEX2 tags to the endogenous KAHRP protein has no discernible impact on knob production or function in cytoadherence.

Proteomic identification of proteins biotinylated by KAHRP-FLAG-APEX2
Biotinylated proteins were enriched using streptavidin magnetic beads from triplicate samples from KAHRP-FLAG-APEX2 line, with material purified in parallel from both the BP+/H 2 O 2 + condition and the BP−/H 2 O 2 + condition to act as a negative control.Proteins were eluted from the beads, precipitated, alkylated and digested with trypsin as described in the Methods, and subjected to LC-MS/MS using an Orbitrap mass analyser.Proteins were identified by searching peptide sequences against the 3D7 proteome, with H. sapiens erythrocyte databank.40,327 peptides were identified in the KAHRP-FLAG-APEX2 BP+/H 2 O 2 + condition and 35,845 peptides were identified in the KAHRP-FLAG-APEX2 BP−/H 2 O 2 + condition (Table S1).The high number of peptides identified in the pull-downs from both conditions does not appear to correlate with the Western blotting (Fig. 2A) and IFA results (Fig. 3B), which both indicate that biotinylated proteins are present at much higher abundance in the BP+/H 2 O 2 + condition than the BP−/H 2 O 2 + condition.However, this is likely due to non-specific purification of non-biotinylated proteins during enrichment.Although the affinity between streptavidin and biotin substrate is strong 41,42 , theoretically favouring highly specific enrichment, nonspecific binding of non-biotinylated proteins to streptavidin beads is a known concern, mostly because of the amount and composition of the streptavidin coated beads used for the protocol 43 .
Given these potential sources of background caused by non-biotinylated proteins being enriched along with biotinylated proteins, we took a deliberately conservative and stringent approach to minimise the risk of false positives.We only considered proteins as potential KAHRP-FLAG-APEX2 interactors if we could detect that protein from at least 2 different peptides, where at least one must be a unique proteotypic peptide 44 that www.nature.com/scientificreports/contains a variable biotin-phenol (+ 361.14601) modification on Tyr residues, confirming that it was definitively biotinylated.We considered only unique biotinylated peptides by which we could assess the signature of the biotin-phenol fragmented ions on the mass spectra as already reported elsewhere 42 .This analysis identified a total of 42 non-redundant biotinylated peptides that were found exclusively in the KAHRP-FLAG-APEX2 BP+/ H 2 O 2 + condition.These peptides corresponded to 8 H. sapiens erythrocyte proteins and 22 P. falciparum proteins, including KAHRP-FLAG-APEX2 (Table S2).
Remarkably, many proteins observed localizing at MC by IFA or proteomic analyses in previous studies were found biotinylated in our dataset.Among these proteins are ring exported protein-1 (REX1) 45 , ring exported protein-2 (REX2) 46 , skeleton-binding protein 1 (SBP1) 47 , early transcribed membrane protein 10.2 (ETRAMP10.2) 48, small exported membrane protein (SEMP1) 49 and parasite-infected erythrocyte surface protein (PIESP2) 48 .It is also noteworthy to mention that host proteins were also tagged by KAHRP-FLAG-APEX2.Among these proteins are α/β-spectrin, Protein 4.1, glycophorin C and band 3.These proteins are intimately associated with RBC cytoskeleton, which could explain their labelling considering KAHRP as main knob constituent 17 (Table 1).Once KAHRP is also transported through MC 47 , it is not at all surprising that other MC proteins may interact with KAHRP at some time point during their export into iRBC membrane.Thus, this study remarks the first report of such direct interactions during in vitro schizogony in P. falciparum development.
Overall, a total of 6 and 12 of the 22 potential parasite KAHRP-interacting proteins were predicted to be secreted by classical or non-classical pathways by SignalP and SecretomeP, respectively.Moreover, 15 and 16 of the 22 potential parasite KAHRP-interacting proteins were predicted to contain at least one transmembrane protein domain by TMHMM and Phobius, respectively (Table S3).Altogether, 21 proteins are predicted to have an extracellular or cell surface localization corresponding to 95% of the 22 potential parasite KAHRP interactors identified.Only protein histone H4 (PF3D7_1105000) was not predicted to be exported by any algorithm used (Table 1).
These 22 potential parasite KAHRP-interacting proteins were also GO-categorized in PlasmoDB (https:// plasm odb.org/ plasmo/ app) to assess the "Cellular Compartment" ontology terms.A total of 24 GO terms for "Cellular Compartment" were returned from our data (Table S4).A significant enrichment was found in the expected GO terms.The most overrepresented GO terms statistically significant in the WordCloud and Bubble plot were related to the host environment (Fig. 5).Together, these bioinformatic approaches establish that the majority of the parasite proteins identified as candidate KAHRP interactors were secreted, exported and/or associated with membranes.

Discussion
In this work, we have used APEX2-based proximity biotin-labelling and shotgun proteomics to identify proteins that are candidate interactors of P. falciparum KAHRP, the critical component of the knobs that form in the iRBC membrane and play a key role in cytoadherence.The APEX2 promiscuous biotinylation domain was integrated into the P. falciparum genome in-frame with the endogenous KAHRP protein, and electron microscopy and cytoadherence assays both detected no effect of the FLAG-APEX2 tag on either knob formation or function (Fig. 4 and Fig. S3), suggesting that KAHRP remained fully functional.After labelling assays, Western blotting confirmed that a set of proteins were biotinylated in this line only when the substrate BP was added, followed by H 2 O 2 to allow oxidation (Fig. 2A).IFA confirmed that these biotinylated proteins were predominantly found in subcellular structures within the iRBC (Fig. 3C).By contrast, in the FLAG-APEX2 line with episomally expressed APEX2 that is not fused to any endogenous protein and hence is cytosolic, the APEX2 was active (Fig. S1) but the localisation of biotinylated proteins were intracellular (Fig. S2).No labelling was also detected in a wildtype control line (Fig. S1).Based on these data, APEX2 has significant promise to identify interacting proteins in P. falciparum.
Despite these clear results from Western blotting and IFA, enrichment of biotinylated proteins by streptavidin beads followed by identification using mass spectrometry revealed high levels of non-specific background in both conditions of the KAHRP-FLAG-APEX2 transfectant.It was therefore expected that there would be some level of non-specific background detectable by mass spectrometry and not by Western blotting/IFA, but there is no question that the level of background in the mass spectrometry data was a surprise.There is a consensus that the use of more streptavidin beads than necessary can increase the background by providing more surface area for nonspecific binders.Elution is also a bottleneck, specifically the difficulty of detaching biotinylated proteins from streptavidin, given the interaction is exceedingly strong 42,43,53 .
Given this significant issue with background, we applied highly stringent filters, assigning proteins as potential KAHRP interactors only if they were identified by two peptides, with at least one being unique containing a biotinylated Tyr, in at least one of the test replicates, and in none of the control replicates.From these unique biotinylated peptides, an additional criteria were employed: the presence of MS/MS spectral data of fragmentation  2 Prediction performed in the web tool "identify genes based on exported protein" from PlasmoDB (https:// plasm odb.org/ plasmo/ app/ search/ trans cript/ Genes ByExp ortPr edict ion). 3Prediction performed in the web tool "identify genes based on protein motif pattern" from PlasmoDB (https:// plasm odb.org/ plasmo/ app/ search/ trans cript/ Genes ByMot ifSea rch).The search was made by using PEXEL motif (RxLx[E/Q/D]) previously established elsewhere 12,52,55 . 4Prediction performed in the SignalP algorithm (https:// servi ces.healt htech.dtu.dk/ servi ces/ Signa lP-5.0/). 5 Prediction performed in the SecretomeP algorithm (https:// servi ces.healt htech.dtu.dk/ servi ces/ Secre tomeP-2.0/). 6Prediction performed in the TMHMM algorithm (https:// servi ces.healt htech.dtu.dk/ servi ces/ TMHMM-2.0/). 7Prediction performed in the Phobius algorithm (https:// phobi us.sbc.su..This conservative and stringent approach identified with high confidence only 42 exclusive non-redundant biotinylated peptides from KAHRP-FLAG-APEX2 BP+/H 2 O 2 + condition, representing a total of 22 parasite and 8 host proteins.It is important to be clear on the limitations and outlines of this dataset.Some interacting proteins may have biotinylation sites that are inaccessible, either because their coiled three-dimensional structure means that the tyrosine residues facing inwards, or they are membrane proteins with little of their sequence exposed to APEX2 activity.Moreover, some proteins that are more transient KAHRP partners but were not in the vicinity of KAHRP when the 'snapshot' of the APEX2-based biotinylation was carried out may be missing. These caveats emphasise that there are false negatives, but there are multiple reasons to be confident that our dataset has captured true KAHRP interactors.To reach the host cell membrane, many parasite proteins exported across the parasitophorous vacuole membrane towards MC need to be processed by PTEX machinery 51,54 .Proteins exported by this mechanism contain a PEXEL motif at N-terminal end 12,55 .KAHRP possesses a PEXEL motif and is processed by this mechanism 12,52 , and transiently carried over into MC 47 .Among our data, beyond KAHRP, several other proteins identified as potential KAHRP interactors also have a PEXEL motif: PTP4 and 5, GBP130, MESA, PIESP2, PHISTa, and Plasmodium exported protein (PF3D7_1353100) (Table 1) 11,12 .Although we cannot establish whether these proteins were tagged by KAHRP-FLAG-APEX2 before, during or after translocation through PTEX machinery, the presence of proteins widely known to be exported through this machinery increases confidence in the hits identified in our dataset.www.nature.com/scientificreports/KAHRP-FLAG-APEX2 confirmed by IFA to be localized in punctate structures (likely MC), as were biotinylated proteins catalysed by it (Fig. 3A).As mentioned already, several of the exported proteins we identified as putative KAHRP interactors are known to be found in the MC, such as REX1 45 , REX2 46 , SBP1 56 , ETRAMP10.2 48, SEMP1 49 and PIESP2 48 (Table 1).Interestingly, all these aforementioned proteins are characterized as PEXEL-Negative Exported Proteins (PNEPs) 57 .Given KAHRP is a true PEXEL-exported protein 12,52 , it is possible that labelling of these PNEPs indicates that some export PEXEL proteins and PNEPs are shared after PEXEL proteins have exited from the PTEX machinery.Some of the proteins identified in our dataset, such as REX1 45 , SBP1 56 and; PTP4 and 5 11 , are already known to be involved in PfEMP1 trafficking.PfEMP1 is often associated with KAHRP, supporting knob formation and/or cytoadherence to the host endothelium 18,47 .Some reports have indicated that KAHRP and PfEMP1 could interact with each other 58,59 , but this statement has been controversial.Structurally, PfEMP1 is a transmembrane protein found at the MC periphery prior redistribution to the iRBC membrane 35 .Moreover, the intracellular portion ATS (acidic terminal segment) at C-termini from PfEMP1, supposedly responsible to create mechanical connections once in the iRBC membrane, does not bind to KAHRP 60 .These topological and biochemical features of PfEMP1 would be reasonable to explain its absence in our dataset, once KAHRP-FLAG-APEX2 would not biotinylate it.
Other structures have been implicated in parasite protein trafficking within the infected erythrocyte, such as transient J-dots/Maurer's clefts (JAM) complex trafficking 61 .One potential KAHRP interactor identified in our data, the Plasmodium exported protein (PF3D7_0702500), has been identified as resident both in the MC and also in the JAM complex 61 .In addition, KAHRP has also been observed in proteomic analysis of extracellular vesicles (EVs) released from ring-stage iRBCs, as have other putative KAHRP interactors found in our dataset such as GBP130, histone H4 and MESA 62 .
Several host proteins were also confirmed to be biotinylated in our dataset possibly concentrated in assembled knobs (Table S1).For instance, KAHRP was already reported to interact with domains 10-14 of β-spectrin, proximal to the spectrin-ankyrin complex 16 , and N-5 fragment (repeat 4) of α-spectrin 63 .Both α and β-spectrin were found biotinylated in our dataset (Table S1).It is also known that spectrin interact with Protein 4.1 64 , and Protein 4.1 is also biotinylated by KAHRP-FLAG-APEX2 in our data set, suggesting a proximity tagging that includes the 4.1-spectrin-KAHRP network in knobs and adjoining erythrocyte cytoskeleton.In addition, other P. falciparum proteins that are biotinylated in our findings have previously been shown to interact with Protein 4.1, specifically Plasmodium exported protein (PHISTa, PF3D7_0402000) 65 and mature parasite infected erythrocyte surface antigen (MESA) 66,67 .Two additional erythrocyte proteins, glycophorin C and band 3, which are known to form a direct or indirect bridge with Protein 4.1 and spectrin, respectively 68,69 , were also biotinylated by KAHRP-FLAG-APEX2.These findings confirm an intimate relation between KAHRP-FLAG-APEX2 and cytoskeleton proteins from host, corroborating KAHRP location and knob architecture in P. falciparum-infected erythrocytes.
In summary, while the biotin-based enrichment used clearly had issues of non-specific binding, our stringent protein identification approach established a list of high confidence list of potential KAHRP interacting proteins that include multiple known exported proteins, as well as host proteins that are known to be enriched in knobs.A schematic representation of how these proteins could interact in spatial and temporal context with KAHRP, either stably (for example by co-assembly into knobs) or transiently (by co-export using different cellular pathways) is proposed in Fig. 6.

Concluding remarks
Protein export in malaria parasites is undoubtedly complex, and is still a field of intense exploration across Plasmodium species.Maurer's Clefts have been widely studied and viewed as a primary structure involved in protein export 12,13,[33][34][35]70 , but recent reports have shown that alternative mechanisms exist 61,62,71,72 . A etter understanding of the protein composition of these pathways is needed, and APEX2 and BioID strategies to establish protein proximity represent technological breakthroughs that could help with this 19,[28][29][30] .Tagging endogenous KAHRP with APEX2 resulted in the biotinylation of parasite proteins already described as exported into the RBC, suggesting that they are neighbouring KAHRP in a given time point.Among our dataset there are likely both direct and transient interactors with KAHRP, which may help to give a broader comprehension in protein export. The study aso clearly identifies the methodological challenges concerning biotinylated protein/ peptide elution in these proximity-tagging approaches 42,43,53,73,74 .Overall, our work has showed for the first time that APEX2 technology can be used for tagging a protein target in P. falciparum during the erythrocyte cycle and that while it has broad potential, rigorous controls and stringent analysis are essential to interpretation.

P. falciparum culture conditions
P. falciparum strain 3D7 parasites were originally obtained from Malaria Research and Reference Reagent Resource Centre (available at: www.mr4.org) and were continuously maintained in O + human erythrocytes as previously described 75 .All methods were carried out by relevant guidelines and regulations.The use of erythrocytes from human donors for parasite culture was approved by the Ethics Committee of NHS Cambridgeshire 4 Research (15/EE/0253) and all donors supplied written informed consent.

Transfection of parasites
For the transfection in P. falciparum, 50 µg of each plasmid was co-precipitated using 3 volumes (v/v) of ethanol and 10% (v/v) of the initial volume of DNA solution of 3 M sodium acetate pH 5.2.The DNA pellet was resuspended and mixed in 100 µL of buffer P3 (Lonza) with 4 µL ATP (625 mM).A total of 100 µL of ring stage-iRBCs with a parasitemia higher than 10% were pelleted and washed in cold sterile cytomix 81 .The washed iRBCs were resuspended in the cytomix containing both DNA plasmids and electroporated with Lonza Nucleofector 4D using programme P3/CM-150 82 .After 3 weeks of drug pressure using 1.25 nM of WR99210 (Jacobus Pharmaceuticals Company, Inc.) for pCC1 selection and 1.5 µM of DSM-1 for pDC2 selection 82 , the Figure 6.Diagram of potential cellular locations for interaction between KAHRP and parasite and host cell partners during protein export.Based on our proteomic data, APEX2-tagged KAHRP has biotinylated a set of proteins.Different trafficking routes throughout iRBC, including translocation via the PTEX complex and possibly alternative export pathways (e.g.PNEP proteins) are proposed to be used by KAHRP until its destination at knobs under the RBC membrane.Identified biotinylated proteins are labelled by a red circle with the letter B. PfEMP1, the main partner of KAHRP is described as a periphery protein found in the MC and its ATS domain does not interact with KAHRP.These topological features probably hampered APEX2 biotinylation.Other MC proteins, such as SBP1, PIESP2, REX1, REX2, SEMP1 and ETRAMP10.2were found biotinylated in this study in agreement with the literature.Moreover, host proteins such as α/β-spectrin, protein 4.1, glycophorin and band 3 that compose cytoskeleton erythrocyte proteins were also biotinylated by KAHRP-APEX2, confirming the involvement of these proteins on the knob architecture.It is known that MC and J-dots comprise the JAM trafficking complex.One protein from our dataset found in this trafficking hub connecting these structures is the Plasmodium exported protein (PF3D7_0702500).Of special note, KAHRP and other proteins, such as GBP130, histone H4 and MESA, which were also found in our dataset as biotinylated, were reported to be in extracellular vesicles from ring-stage parasites, suggesting that protein export mechanisms in P. falciparum is much more complex than presumed. Vol.:(0123456789)

Biotinylation assay
Biotinylation assays were carried out using both wildtype control strains, and the KAHRP-APEX2 tagged line.Parasites were routinely synchronized using sorbitol treatment.Before biotinylation around 2 mL of RBCs at 5% parasitemia (corresponding to 1 × 10 9 iRBCs) of predominantly late stages (trophozoites/schizonts) were enriched by centrifugation over 63% (v/v) isotonic Percoll cushion (Percoll Plus 17-5445-01, GE Healthcare), with the interface, which contains primarily late-stage parasites, isolated.This parasite fraction was then divided in equal volumes between the different test conditions in order to have the same cell number in all conditions.The enriched parasites were resuspended in complete RPMI medium without phenol red, supplemented with or without 250 µM biotin-phenol (Iris Biotech, CDX-B0270), and incubated for 30 min at 37 ºC, 5% CO 2 to allow biotinylation to occur.After the incubation period each condition was split in two, with half having 1 mM H 2 O 2 added (final concentration; GE Healthcare) for 1 min, and half not.Samples from a total of four conditions were therefore generated-in the presence or absence of biotin-phenol (BP), and each one with or without H 2 O 2 .
Immediately after H 2 O 2 addition the reactions were quenched with an antioxidant solution containing 10 mM sodium azide (Sigma-Aldrich), 10 mM sodium ascorbate (Sigma-Aldrich) and 5 mM of Trolox (Sigma-Aldrich) in PBS.A fraction of the iRBCs were pelleted to make blood smears used for immunofluorescence assays.The remaining iRBCs were lysed with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS containing EDTA-free protease inhibitor cocktail (cOmplete™, Roche), and the pellets (which contain both intact parasite and membrane material from the iRBC including Maurer's Clefts) were frozen for subsequent use in Western blots and proteomics.Material from all four conditions were used in Western Blots, but only two were then used for subsequent pulldown of biotinylated proteins and proteomic analysis: BP+/H 2 O 2 + and BP−/H 2 O 2 +.

Immunofluorescence assays
Smears from KAHRP-APEX2 or WT parasites enriched for late stages were prepared on glass slides and dried for at least 30 min before fixing with 4% (v/v) paraformaldehyde (Sigma-Aldrich) for 20 min and washing once with PBS.Cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in PBS and blocked for 3 h at 8 °C using 3% (w/v) BSA in PBS.To identify the location of the KAHRP-APEX2 fusion, the parasites were probed with mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich, F3165; 1:800 dilution) for 1 h at room temperature in blocking solution followed by staining with an Alexa Fluor 555-conjugated anti-mouse IgG goat antibody (Invitrogen™, A21424; 1:800 dilution).To identify proteins biotinylated following the biotinylation assay, slides were co-probed with Alexa-Fluor 488-conjugated streptavidin (Invitrogen™, S11223; 1:800 dilution) and IgG rabbit antibodies directly raised against PfMAHRP or PfERD2 (MRA; 1:200 dilution for both) for 1 h at room temperature in blocking solution.After three washes in PBS, cells were incubated with Alexa-Fluor 546-conjugated anti-rabbit IgG donkey antibodies (Invitrogen™, A10040; 1:800 dilution).After a final wash, the slides were mounted with anti-fade mounting medium with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes).Confocal images were acquired with a LSM510 laser scanning confocal microscope (Zeiss).

Scanning electron microscopy (SEM)
Trofozoites and schizonts from both 3D7 and KAHRP-FLAG-APEX2 lines were enriched by using Percoll cushion 63% (v/v) (Percoll Plus  15,83 .Cells were then washed with PBS and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h.After fixation, cells were conditioned in 0.1 M sodium cacodylate.Following the procedure, cells were fixed in contrast solution (0.1% (w/v) OsO 4 in 0.1 M sodium cacodylate buffer) at room temperature.After fixation, cells were washed with distilled water.Coverslips were prepared with poly-l-lysine to attach trophozoites/schizonts-iRBCs, followed by acetone gradient dehydration (50%, 70%, 90% and 100%).Critical drying of samples was performed by using CPD 030 critical point dryer (BALZERS, USA).Dried samples were mounted on suitable sample holders using conductive adhesive and taken to the SCD 500 metalizer (Leica, Germany).All analyses were performed in the JSM-7001F (JEOL, Japan) SEM microscope, and the images were collected at an increase of 10.000 × with 15.0 kv.

Proteomic analysis
After confirmation by streptavidin-HRP blot that the KAHRP-FLAG-APEX2 line was producing biotinylated proteins, independent biological triplicate samples from the conditions BP+/H 2 O 2 + and BP−/H 2 O 2 + were obtained, with the same independent triplicates samples from the same conditions also obtained from the wildtype 3D7 parasite strain as an additional control.All samples were enriched using streptavidin beads as described above, and eluted material precipitated with 4 volumes of cold acetone to the sample in a proportion of 80:20.The protein pellet was washed twice with cold ethanol 70% (v/v), followed with the air drying in the fume hood [84][85][86] .The protein pellet was then resuspended in 25 mM triethylammonium bicarbonate buffer (TEAB, Sigma-Aldrich), reduced with 5 mM dithiothreitol (GE Healthcare) for 30 min, at 55 ºC, followed by alkylation of disulfide bonds with 14 mM iodoacetamide (Sigma-Aldrich) for 40 min in the dark, at room temperature.Next, the sample was digested with trypsin gold (Promega) at 37 ºC, for 18 h, then desalted using a homemade desalting system of packed P-200 C18 columns (3 M™ Empore™) and Poros OligoR3 resin (Applied Biosystems™) 87 .The eluted peptides were dried in SpeedVac centrifuge (Eppendorf™) using LoBind® tubes (Eppendorf™) and stocked at -20 ºC until the day of usage.Liquid chromatography (LC) was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled online to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).Peptides were trapped on a C18 column (75 μm inner diameter × 2 cm; nanoViper Acclaim PepMap™ 100, Thermo Scientific) with buffer A (2/98 MeCN/H 2 O in 0.1% formic acid) at a flow rate of 3.0 µL/min over 4 min.Separation was performed on a 50 cm × 75 μm C18 column (nanoViper Acclaim PepMap™ RSLC, 2 μm, 100 Å, Thermo Scientific) regulated to a temperature of 40 °C with a linear gradient of 3% to 29% buffer B (B: 100% MeCN in 0.1% formic acid, A: 100% H 2 O in 0.1% formic acid) at a flow rate of 300 nL/min over 91 min.MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 375-1500 with a resolution of 120,000 at m/z 200.The top 20 most intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation and a resolution of 15,000 with the auto gain control (AGC) target set to 100%.We selected ions with charge state from 2+ to 6+ for screening.Normalized collision energy (NCE) was set at 30 and the dynamic exclusion of 40 s.
For identification, the data were searched against the P. falciparum strain 3D7 proteome databank (Proteome UP000001450, from August, 2021) with 5,456 entries downloaded from Uniprot (https:// www.unipr ot.org/).To this file, 438 entries that contained "red blood cells", "hematopoiesis" and "erythrocytes" associated key words from the Homo sapiens proteome databank (Proteome UP000005640, from August, 2021) were manually added to identify potential erythrocyte proteins, and the KAHRP-FLAG-APEX2 chimeric sequence was also manually added.Enzyme specificity was set to trypsin and a maximum of two miss cleavages sites were allowed.Carbamidomethylation of cysteine (+ 57.02) was set as fixed modification.Oxidized methionine (+ 15.99), biotinyl-tyramide of tyrosine residues (+ 361.14601) and N-terminal acetylation (+ 42.01) were set as variable modifications 42 .Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS/MS peaks.The resulting files were further processed using PEAKS Studio 7.0.FDR was set to 0.01 at the peptide level for the whole study.To determine a protein as "biotinylated", a manual selection of 2 peptides, with at least one being unique, was used 44,88 .All identified proteins must have at least one unique peptide with a variable biotin-phenol (+ 361.14601) modification on Tyr residues in at least one of the test replicates.To ensure that all unique biotinylated peptides used to identify KAHRP candidate interactors are indeed tagged by biotinylation, a manual screening was performed to detect through MS/MS spectra data from fragmentation ions signature of dehydrobiotin (m/z 227.08), immonium bpY (m/z 497.22) and/or immonium bpY with loss of -NH 3 (m/z 480.195), as described previously 42 .All intensities of these fragmentation ions were also listed.

Bioinformatic analysis
Proteins predicted to contain the export PEXEL motif (RxLx[EQD]) were obtained from PlasmoDB (https:// plasm odb.org/ plasmo/ app).Overrepresented Gene Ontology (GO) terms were predicted by GO Cloud tool through PlasmoDB (https:// plasm odb.org/ plasmo/ app) databank.The bubble plots from those enriched GO terms were obtained from R programming language by using ggplot2 package 89 .The potential of identified proteins to be secreted and/or exported to the extracellular environment of all proteins were assessed using the SignalP 90,91 and SecretomeP WebServer 92,93 .For transmembrane protein prediction, TMHMM 94 and Phobius 95 were additionally employed.

Figure 1 .
Figure 1.CRISPR/Cas9 guided knock-in scheme to tag the Plasmodium falciparum kahrp gene with APEX2.(A) A schematic figure illustrating integration of the cassette from the pCC1-KAHRP-FLAG-APEX2 vector into parasite gDNA, resulting in integration of the FLAG tag and APEX2 domain at the 3′ end of the gene, in frame with the KAHRP ORF.(B) PCR validation of cassette integration.Legend: K (KAHRP-FLAG-APEX2) and C-(negative control).

Figure 2 .
Figure 2. Confirmation of chimeric protein expression and biotinylation activity.(A) Ponceau S stained nitrocellulose membrane (left panel), Western-blot (middle panel, anti-FLAG-HRP dilution at 1:800) and streptavidin-blot (right panel, streptavidin-HRP dilution at 1:10,000) of KAHRP-FLAG-APEX2 parasites treated and/or not treated with BP and H 2 O 2 .In all cases, material was generated by saponin lysis of iRBCs, which releases RBC cytosolic contents but retains parasite and exported membrane material, followed by solubilisation of all retained material using RIPA buffer.(B) NuPAGE of material enriched from the KAHRP-FLAG-APEX2 line following biotinylation, lysis with saponin and then RIPA, and purification of biotinylated proteins using streptavidin-coated beads enrichment.Eluted proteins stained by Coomassie-blue were only detected in the material from the BP+/H 2 O 2 + condition (right panel).Legend for all gels/blots: first lane BP−/H 2 O 2 − (not treated with BP or H 2 O 2 ); second lane BP−/H 2 O 2 + (not treated with BP, treated with H 2 O 2 ); third lane BP+/ H 2 O 2 − (treated with BP but not treated with H 2 O 2 ); fourth lane BP+/H 2 O 2 + (treated with both BP and H 2 O 2 ).

Figure 5 .
Figure 5. GO term annotation.(A) GO Cloud obtained from PlasmoDB (https:// plasm odb.org/ plasmo/ app/) corresponding to the "Cellular Component" terms annotated.(B) Bubble plot from top 10 GO terms with most significant p-values from panel (A).

Table 1 .
List of the candidate neighbouring parasite protein partners of the knob-associated histidine rich protein during schizogony in P. falciparum.*This protein ID refers to the chimeric sequence of KAHRP-FLAG-APEX2. 1 Gene ID obtained from PlasmoDB database (https:// plasm odb.org/ plasmo/ app/).